Figure 1.
Strain HF13 is an lpxL1 mutant.
(A) Mass spectrum of HF13 lipid A. The highest peak (1653.6) corresponds to penta-acylated lipid A with two phosphate groups and one phosphoethanolamine (PEA); the second peak (1530.4) corresponds to penta-acytlated lipid A with two phosphate groups without PEA. (B) Depiction of N. meningitidis wild-type lipid A. The acyl chain that is added by LpxL1 is indicated.
Figure 2.
Presentation of all lpxL1 mutations.
The lpxL1 gene sequence of strain MC58 is shown including the different types of mutations and their positions in the gene found among isolates from patients. Each type of mutation is indicated with a different color. An ‘_’ indicates a nucleotide that is deleted in the mutant strain, pink indicates an insertion element (type I and II mutations), yellow indicates a deletion of a guanine in a stretch of five guanines (type III mutation), green indicates a deletion of an adenosine in a stretch of five adenosines (type IV mutation), light blue indicates a deletion of an adenosine in a stretch of seven adenosines (type V mutation), dark blue indicates an insertion of an adenosine in a stretch of seven adenosines (type V mutation), and the sequences that are highlighted in red or gray are deleted in the mutant strain (type VI and VII mutations respectively).
Table 1.
List of all lpxL1 mutant strains.
Figure 3.
Comparison of IL-6 induction in MM-6 cells between wild-type strains and lpxL1 mutants.
MM-6 cells were stimulated for 18 h with titrations of indicated strains and IL-6 in supernatant was quantified with ELISA. H44/76 is a wild-type strain, L8 lpxL1 is a constructed lpxL1 mutant, pLAK33 is an LPS-deficient mutant, and all other strains are spontaneous lpxL1 mutants. Results of one representative experiment of three independent experiments are shown. Error bars indicate S.E.M. of triplicates.
Figure 4.
Comparison between wild-type strains and lpxL1 mutants of IL-8 induction in HEK293 cells transfected with human TLR4.
HEK293 cells transfected with human TLR4, CD14, and MD-2 were stimulated for 18 h with titrations of indicated strains and IL-8 in supernatant was quantified with ELISA. H44/76 is a wild-type strain, L8 lpxL1 is a constructed lpxL1 mutant, pLAK33 is an LPS-deficient mutant, and all other strains are spontaneous lpxL1 mutants. Results of one representative experiment of three independent experiments are shown. Error bars indicate S.E.M. of triplicates.
Figure 5.
Comparison between wild-type strains and lpxL1 mutants in pro-inflammatory cytokine induction in PBMCs.
PBMCs from three different donors were stimulated with titrations of the indicated strains and IL-6, TNF-α, and IL-1β were quantified in the supernatant 18 h after stimulation. H44/76 is a wild-type strain, L8 lpxL1 is a constructed lpxL1 mutant, pLAK33 is an LPS-deficient mutant, and all other strains are spontaneous lpxL1 mutants. Results of one representative experiment of two independent experiments are shown. Error bars indicate S.E.M. of triplicates.
Figure 6.
Clinical correlate of lpxL1 mutations in meningococcal meningitis.
(A) Frequency of rash in patients presenting with meningitis infected by lpxL1 wild-type and mutant strains. (B,C) Platelet counts on admission for lpxL1 wild-type and mutant strains (B) and patients presenting with and without rash (C). Horizontal bars reflect medians. The Mann-Whitney U test was used to identify differences between groups in continuous variables, and dichotomous variables were compared by the chi-square or Fisher exact test.
Table 2.
Clinical features of 254 adults with meningococcal meningitis due to lpxL1 mutant and wild-type strains.